Although it is well known that women are more susceptible to the toxic effects of ethanol (EtOH) than men, much less is known about the molecular mechanisms underlying alcohol toxicity in women especially as relates to bone. Alcohol abuse during early adulthood results in impaired bone growth and in the U.S.A. approximately 20% of women age 18-30 (4.4 million) binge drink. A resulting reduction in peak bone mass would predispose women to osteoporosis in later life. The molecular mechanisms underlying the toxic effects of EtOH on bone remain poorly understood. EtOH inhibits bone formation. Bone loss in female rats and mice fed EtOH is blocked by dietary antioxidants including N-acetylcysteine and vitamin E and by DPI an inhibitor of NADPH oxidase (NOX) enzymes. However, effects of EtOH to inhibit osteoblastogenesis and stimulate bone marrow adiposity were not blocked in p47phox -/- mice lacking active NOX1/2. We hypothesize that another source of reactive oxygen species (ROS) mediates the effects of EtOH on bone formation. Ex-vivo bone marrow cultures from NOX 4 -/- mice will be utilized to test the hypothesis that NOX4 mediates the effects EtOH-induced inhibition of mesenchymal stem cell self-renewal and differentiation into osteoblasts or adipocytes. A mouse liquid diet model will be used to test this hypothesis in vivo in mice with cell specific ablation of NOX4 in osteoblast precursors (Prx-1-Cre-lox mice). Alternatively, the role of mitochondrial derived ROS in EtOH inhibition of bone formation will be tested using the mitochondrial ROS inhibitor mitoTEMPO. At higher concentrations characteristic of binge drinking, EtOH can also increase bone resorption via induction of RANKL, a member of the TNF family expressed primarily in osteocytes, which signals through the receptor RANK to stimulate osteoclastogenesis. The role of NOX derived ROS in EtOH-induced RANKL production from osteocytes and other effects of EtOH on osteocyte function and cortical bone morphology will be determined in vivo in mice lacking NOX4 or with inactive NOX1/2 in osteocytes (NOX4 and rac-1 Dmp-1 Cere-lox mice) fed EtOH via liquid diet and in an EtOH binge drinking model. In addition, the role of excess NADH produced during EtOH metabolism in NOX activation will be tested in bone cells in vitro.